Objectives: Vitamin D is one of the major hormones involved in the metabolism of calcium (Ca) and phosphorus (P). In the present study, we aimed to determine the analytical performance of the immunoassay method used for determining plasma 25-hydroxyvitamin D [25(OH)D] levels in routine clinical practice in laboratories. Methods: Venous blood samples were collected from 156 patients for the comparisons and were analyzed with Siemens ADVIA Centaur XPT, the Roche Cobas 6,000's module e601, Abbott Architect i2000, and the liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results: The four methods were analyzed and compared through the Passing-Bablok regression for 25(OH)D, and the highest correlation was found at LC-MS/MS and Cobas 6,000's module e601 (r=0.799), LC-MS/MS/Abbott Architect i2000, and LC-MS/MS/Siemens ADVIA Centaur XPT as r=0.736, 0.721, respectively. The correlation coefficient was found between Abbott Architect i2000 with Roche Cobas e601 and Siemens ADVIA Centaur XPT as r=0.934 and r=0.907, respectively. Also, the correlation coefficient was found between Roche Cobas e601 and Siemens ADVIA Centaur XPT as r=0.906. Conclusions: The Roche Cobas assay showed better performance, compared with the other assays. Based on our findings, the chemiluminescence methods in automated systems seem to be expedient.